The four-way DNA junction known as the Holliday junction (HJ) figures prominently in DNA recombination, replication and repair;it is the central intermediate in both homologous and site-specific recombination, where genetic, biochemical and structural studies of the HJ have been particularly fruitful. We have been studying formation, properties, and resolution of this recombination intermediate in the ? phage recombination pathway, a paradigm for a large family of site-specific recombinases that administers a wide range of functions in prokaryotes, eukaryotes, and archaea. Recombinases of this family catalyze rearrangements between DNA sequences (called att sites in the ? system) with very little homology to each other and have the ability to generate and resolve HJs without the input of energy. Experiments in this proposal grow out of a large body of biochemical, genetic, and structural data that afford insights into how HJs are generated and resolved by the ? phage-encoded Int protein, which is also a model for the large subset of virally-encoded family members that are heterobivalent DNA binding proteins. The four specific aims address questions growing out of results obtained during the previous project period and/or of long standing in the field. They are: 1) to test the functional implications of the crystal structure of HJ complexed with Int and arm-type oligonucleotides (HJ-Int-Arm complex);2) to determine which chemical and/or conformational steps are required to form a stable synaptic complex;3) to determine the rate limiting step in formation of the HJ;4) to determine the numerical distribution of attL-bound and attR-bound Integrase subunits that lead to successful synaptic events and the subsequent formation of stable Holliday junction. Aims 1-3 depend upon approaches developed and utilized during the previous project period. Aim 4 involves a logical extension of some of those techniques. Several of the questions posed in aims 2-4 would be extremely difficult to answer using ensemble biochemistry since many of the relevant intermediates are transient and/or difficult to verify as being on-pathway events. We propose to address these questions using logical extensions of our investment in single molecule approaches to studying site-specific recombination (something which is thus far unique in the field). The four-way DNA junction known as the Holliday junction figures prominently in DNA recombination, replication and repair. We have been studying formation and resolution of this recombination intermediate in the ? phage-encoded recombination pathway, a paradigm for a large family of site-specific recombinases that is ubiquitous in nature and administers a wide range of functions, many of which figure prominently in various aspects of health-related issues.